Phosphorylation regulates integration of the yeast Ty5 retrotransposon into heterochromatin

The yeast Ty5 retrotransposon preferentially integrates into heterochromatin at the telomeres and silent mating loci. Target specificity is mediated by a small domain of Ty5 integrase (the targeting domain, TD), which interacts with the heterochromatin protein Sir4 and tethers the integration complex to target sites. Here we demonstrate that TD is phosphorylated and that phosphorylation is required for interaction with Sir4. The yeast cell, therefore, through posttranslational modification, controls Ty5's mutagenic potential: when TD is phosphorylated, insertions occur in gene-poor heterochromatin, thereby minimizing deleterious consequences of transposition; however, in the absence of phosphorylation, Ty5 integrates throughout the genome, frequently causing mutations. TD phosphorylation is reduced under stress conditions, specifically starvation for amino acids, nitrogen, or fermentable carbon. This suggests that Ty5 target specificity changes in response to nutrient availability and is consistent with McClintock's hypothesis that mobile elements restructure host genomes as an adaptive response to environmental challenge.     

Yeast Silent Mating Type Loci Form Heterochromatic Clusters through Silencer Protein-Dependent Long-Range Interactions

The organization of eukaryotic genomes is characterized by the presence of distinct euchromatic and heterochromatic sub-nuclear compartments. In Saccharomyces cerevisiae heterochromatic loci, including telomeres and silent mating type loci, form clusters at the nuclear periphery. We have employed live cell 3-D imaging and chromosome conformation capture (3C) to determine the contribution of nuclear positioning and heterochromatic factors in mediating associations of the silent mating type loci. We identify specific long-range interactions between HML and HMR that are dependent upon silencing proteins Sir2p, Sir3p, and Sir4p as well as Sir1p and Esc2p, two proteins involved in establishment of silencing. Although clustering of these loci frequently occurs near the nuclear periphery, colocalization can occur equally at more internal positions and is not affected in strains deleted for membrane anchoring proteins yKu70p and Esc1p. In addition, appropriate nucleosome assembly plays a role, as deletion of ASF1 or combined disruption of the CAF-1 and HIR complexes abolishes the HML-HMR interaction. Further, silencer proteins are required for clustering, but complete loss of clustering in asf1 and esc2 mutants had only minor effects on silencing. Our results indicate that formation of heterochromatic clusters depends on correctly assembled heterochromatin at the silent loci and, in addition, identify an Asf1p-, Esc2p-, and Sir1p-dependent step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions.     

Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast

A general feature of the nucleus is the organization of repetitive deoxyribonucleic acid sequences in clusters concentrating silencing factors. In budding yeast, we investigated how telomeres cluster in perinuclear foci associated with the silencing complex Sir2-Sir3-Sir4 and found that Sir3 is limiting for telomere clustering. Sir3 overexpression triggers the grouping of telomeric foci into larger foci that relocalize to the nuclear interior and correlate with more stable silencing in subtelomeric regions. Furthermore, we show that Sir3's ability to mediate telomere clustering can be separated from its role in silencing. Indeed, nonacetylable Sir3, which is unable to spread into subtelomeric regions, can mediate telomere clustering independently of Sir2-Sir4 as long as it is targeted to telomeres by the Rap1 protein. Thus, arrays of Sir3 binding sites at telomeres appeared as the sole requirement to promote trans-interactions between telomeres. We propose that similar mechanisms involving proteins able to oligomerize account for long-range interactions that impact genomic functions in many organisms.     

Stress management: how cells take control of their transposons

In this issue of Molecular Cell, Dai et al. (2007) describe their exciting discovery that, in Saccharomyces cerevisiae, the integrase of retrotransposon Ty5 is phosphorylated, and this modification stabilizes the interaction between integrase and Sir4p that directs integration to heterochromatin.     

Esc1, a nuclear periphery protein required for Sir4-based plasmid anchoring and partitioning

A targeted silencing screen was performed to identify yeast proteins that, when tethered to a telomere, suppress a telomeric silencing defect caused by truncation of Rap1. A previously uncharacterized protein, Esc1 (establishes silent chromatin), was recovered, in addition to well-characterized proteins Rap1, Sir1, and Rad7. Telomeric silencing was slightly decreased in Deltaesc1 mutants, but silencing of the HM loci was unaffected. On the other hand, targeted silencing by various tethered proteins was greatly weakened in Deltaesc1 mutants. Two-hybrid analysis revealed that Esc1 and Sir4 interact via a 34-amino-acid portion of Esc1 (residues 1440 to 1473) and a carboxyl-terminal domain of Sir4 known as PAD4 (residues 950 to 1262). When tethered to DNA, this Sir4 domain confers efficient partitioning to otherwise unstable plasmids and blocks the ability of bound DNA segments to rotate freely in vivo. Here, both phenomena were shown to require ESC1. Sir protein-mediated partitioning of a telomere-based plasmid also required ESC1. Fluorescence microscopy of cells expressing green fluorescent protein (GFP)-Esc1 showed that the protein localized to the nuclear periphery, a region of the nucleus known to be functionally important for silencing. GFP-Esc1 localization, however, was not entirely coincident with telomeres, the nucleolus, or nuclear pore complexes. Our data suggest that Esc1 is a component of a redundant pathway that functions to localize silencing complexes to the nuclear periphery.     

Separation of silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins

In budding yeast, the nuclear periphery forms a subcompartment in which telomeres cluster and SIR proteins concentrate. To identify the proteins that mediate chromatin anchorage to the nuclear envelope, candidates were fused to LexA and targeted to an internal GFP-tagged chromosomal locus. Their ability to shift the locus from a random to a peripheral subnuclear position was monitored in living cells. Using fusions that cannot silence, we identify YKu80 and a 312-aa domain of Sir4 (Sir4(PAD)) as minimal anchoring elements, each able to relocalize an internal chromosomal locus to the nuclear periphery. Sir4(PAD)-mediated tethering requires either the Ku complex or Esc1, an acidic protein that is localized to the inner face of the nuclear envelope even in the absence of Ku, Sir4 or Nup133. Finally, we demonstrate that Ku- and Esc1-dependent pathways mediate natural telomere anchoring in vivo. These data provide the first unambiguous identification of protein interactions that are both necessary and sufficient to localize chromatin to the nuclear envelope.     

Targeting of the yeast Ty5 retrotransposon to silent chromatin is mediated by interactions between integrase and Sir4p

The Ty5 retrotransposons of Saccharomyces cerevisiae integrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR and HML). We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP). The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed. As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin. This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity.     

A nonhistone protein-protein interaction required for assembly of the SIR complex and silent chromatin

Budding yeast silent chromatin, or heterochromatin, is composed of histones and the Sir2, Sir3, and Sir4 proteins. Their assembly into silent chromatin is believed to require the deacetylation of histones by the NAD-dependent deacetylase Sir2 and the subsequent interaction of Sir3 and Sir4 with these hypoacetylated regions of chromatin. Here we explore the role of interactions among the Sir proteins in the assembly of the SIR complex and the formation of silent chromatin. We show that significant fractions of Sir2, Sir3, and Sir4 are associated together in a stable complex. When the assembly of Sir3 into this complex is disrupted by a specific mutation on the surface of the C-terminal coiled-coil domain of Sir4, Sir3 is no longer recruited to chromatin and silencing is disrupted. Because in sir4 mutant cells the association of Sir3 with chromatin is greatly reduced despite the partial Sir2-dependent deacetylation of histones near silencers, we conclude that histone deacetylation is not sufficient for the full recruitment of silencing proteins to chromatin and that Sir-Sir interactions are essential for the assembly of heterochromatin.     

Correct integration of model substrates by Ty1 integrase

The retrovirus-like mobile genetic element of Saccharomyces cerevisiae, Ty1, transposes to new genomic locations via the element-encoded integrase (IN). Here we report that purified recombinant IN catalyzed correct integration of a linear DNA into a supercoiled target plasmid. Ty1 virus-like particles (VLPs) integrated donor DNA more efficiently than IN. VLP and IN-mediated insertions occurred at random sites in the target. Mg(2+) was preferred over Mn(2+) for correct integration, and neither cation enhanced nonspecific nuclease activity of IN. Products consistent with correct integration events were also obtained by Southern analysis. Recombinant IN and VLPs utilized many, but not all, linear donor fragments containing non-Ty1 ends, including a U3 mutation which has been shown to be defective for transposition in vivo. Together, our results suggest that IN is sufficient for Ty1 integration in vitro and IN interacts with exogenous donors less stringently than with endogenous elements.     

A pivotal role of the coiled coil of Sir4

The C terminus of Sir4 forms a coiled-coil structure. The coiled-coil domain is responsible for the dimerization of Sir4 and contains the binding site of Sir3. Structural and biochemical analyses of the Sir4 coiled-coil domain provide important insights into the molecular mechanisms of Sir3-Sir4 interaction and the assembly of a ternary Sir2/Sir3/Sir4 complex that are essential for epigenetic control of gene expression in S. cerevisiae.     

Increase in Ty1 cDNA recombination in yeast sir4 mutant strains at high temperature

Transposition of the Ty1 element of the yeast Saccharomyces cerevisiae is temperature sensitive. We have identified a null allele of the silent information regulator gene SIR4 as a host mutant that allows for transposition at high temperature. We show that the apparent increase in transposition activity in sir4 mutant strains at high temperature is dependent on the RAD52 gene and is thus likely resulting from an increase in Ty1 cDNA recombination, rather than in IN-mediated integration. General cellular recombination is not increased at high temperature, suggesting that the increase in recombination at high temperature in sir4 mutants is specific for Ty1 cDNA. Additionally, this high-temperature Ty1 recombination was found to be dependent on functional Sir2p and Sir3p. We speculate that the increase in recombination seen in sir4 mutants at high temperature may be due to changes in chromatin structure or Ty1 interactions with chromosomal structures resulting in higher recombination rates.     

Dynamic modulation of HIV-1 integrase structure and function by cellular lens epithelium-derived growth factor (LEDGF) protein

The mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra- and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target.     

Heterochromatin formation via recruitment of DNA repair proteins

Heterochromatin formation and nuclear organization are important in gene regulation and genome fidelity. Proteins involved in gene silencing localize to sites of damage and some DNA repair proteins localize to heterochromatin, but the biological importance of these correlations remains unclear. In this study, we examined the role of double-strand-break repair proteins in gene silencing and nuclear organization. We find that the ATM kinase Tel1 and the proteins Mre11 and Esc2 can silence a reporter gene dependent on the Sir, as well as on other repair proteins. Furthermore, these proteins aid in the localization of silenced domains to specific compartments in the nucleus. We identify two distinct mechanisms for repair protein–mediated silencing—via direct and indirect interactions with Sir proteins, as well as by tethering loci to the nuclear periphery. This study reveals previously unknown interactions between repair proteins and silencing proteins and suggests insights into the mechanism underlying genome integrity.